The development of protective or therapeutic HIV vaccines has been hampered by unprecedented challenges, primarily related to the unique properties of the HIV-1 envelope (Env) trimer, a cleverly engineered entry machinery that features an extraordinary assortment of immune-evasion tactics, including antigenic variation, conformational camouflage and heavy glycosylation of all exposed surfaces. Further insights into the complex structure of the HIV-1 Env and its protective shield will thus be critical to guide the design of a protective vaccine. 1) Two tyrosine residues in the V2 domain of HIV-1 gp120 can be sulfated and functionally mimic sulfotyrosines in the CCR5 N-terminal domain. Tyrosine sulfation is a post-translational modification that occurs in approximately 7% of mammalian proteins, including several host proteins that interact with HIV-1 gp120 such as the coreceptors CCR5 and CXCR4, and monoclonal antibodies to the coreceptor-binding site (eg, 412d) and the V2 loop (eg, PG9). We found that two tyrosines in the central region of V2 (Tyr173 and Tyr177) are sulfated and mediate intramolecular interaction with the CCR5-binding site at the base of the V3 loop. In fact, such sulfated tyrosines are structural and functional mimics of two sulfotyrosines in the N-terminus of CCR5, which are essential for the coreceptor function in the process of HIV-1 entry. We found that a tyrosine-sulfated peptide mimetic derived from V2, but not its unsulfated counterpart, interacts with the CCR5-binding site at the base of V3 and adopts a helical configuration analogous to that documented for a CCR5-derived tyrosine-sulfated N-terminal peptide bound to gp120-CD4 complexes. 2) The V2 tyrosines are key constraints in stabilizing the closed, antibody-protected conformation of the HIV-1 Env trimer, thereby providing an important mechanism of immune evasion. By modulating the levels of tyrosine sulfation or by mutagenesis, we demonstrated that the V2 tyrosines serve as critical structural constraints which control the neutralization sensitivity of HIV-1 by shifting the conformational equilibrium of the Env trimer toward the closed, antibody-protected conformation. In fact, enhancement of sulfation reduces the accessibility of the CD4- and coreceptor-binding sites, while inhibition of sulfation or tyrosine mutagenesis has reciprocal effects, favoring the adoption of a more open trimer conformation. Of note, antibodies that preferentially recognize the closed trimer, such as the glycan-dependent anti-V2 antibodies PG9 and PG16, exhibit a reversed pattern with increased binding to the fully sulfated envelope and decreased recognition of the hyposulfated or tyrosine-mutagenized trimer. Strikingly, the pattern of HIV-1 neutralization sensitivity under conditions of increased or decreased tyrosine sulfation precisely mirrors the antigenic profiles. Altogether, these results identify the sulfotyrosine-mediated V2-V3 interaction as a key structural constraint that contributes to stabilizing the native, antibody-protected conformation of the HIV-1 envelope trimer. To investigate whether the sulfotyrosine-mediated V2-V3 interaction could promote HIV-1 immune evasion, we tested the neutralizing capacity of a large panel of sera from HIV-infected patients against HIV-1 pseudotypes carrying either the wild-type or the doubly alanine-substituted (Y173A/Y177A) mutant BaL envelope. The results were dramatic, showing a staggering increase in neutralization sensitivity upon mutation of the two V2 tyrosines, with half-maximal neutralizing titers reaching beyond 1:50,000 for the majority of the sera tested, while the wild-type virus was typically neutralized within the 1:10-1:500 dilution range. These data demonstrate that the V2 tyrosines help to maintain the envelope trimer in its antibody-protected pre-fusion conformation, preventing the binding of abundantly produced host antibodies directed against critical neutralization epitopes such as the CD4- and coreceptor-binding sites. 3) Tyrosine-sulfated peptides derived from the V2 loop are potent and specific inhibitors of HIV-1 entry. To further investigate the structural and functional relevance of tyrosine sulfation in the gp120 V2 loop, we derived short mimetic peptides from the sulfated region of V2 and tested their biological activity. We found that a doubly-sulfated peptide, but not its unsulfated counterpart, binds with nanomolar affinity to gp120 when the glycoprotein is pre-activated with soluble CD4 (sCD4); furthermore, it inhibits binding of a sCD4-treated soluble cleaved gp140 trimer (BG505 SOSIP.664) to CCR5. Accordingly, the peptide specifically blocks HIV-1 entry and fusion by preventing coreceptor utilization. Experiments with singly-sulfated peptides and single alanine mutants of the full-length envelope confirmed the predominant role of Tys177 in mediating V2-V3 interaction. Of note, inhibition occurs on a broad range of HIV-1 isolates with different coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site. In contrast, a CCR5 N-terminus-derived sulfated peptide inhibits selectively CCR5-tropic HIV-1 isolates. The molecular basis for this selectivity, which contrasts with the broad-spectrum activity of the V2-derived peptides, was mapped to a negatively charged residue next to the first sulfotyrosine (Tys10) of CCR5, which conflicts with a negatively charged residue frequently present at position 440 within the coreceptor-binding site of CXCR4-specific envelopes. These data confirm that the sulfated region of V2 is a functional mimic of the CCR5 N-terminus interacting with the coreceptor-binding site at the base of the V3 loop. 4) The CD4 receptor binds to a quaternary surface in the HIV-1 Env trimer. Recently, we discovered that HIV-1 interacts with its main cellular receptor, CD4, through a quaternary surface formed by coalescence of the classic CD4-binding site on one gp120 protomer with a previously unrecognized binding site (that we named CD4-binding site 2, or CD4-BS2) in the inner domain of a neighboring gp120 protomer. Interaction with CD4-BS2 is functionally critical for HIV-1 infectivity as it triggers sequential conformational changes in the Env trimer that lead to the formation/exposure of the coreceptor-binding site and the subsequent progression of the HIV-1 fusogenic process. The discovery of a new CD4-binding site paves the way toward the development of new strategies of treatment and vaccine.